Didemins were isolated from the Caribbean tunicate Trididemnum solidum1. These cyclic depsipeptides possess a variety of biological activities including in vitro and in vivo antiviral, antitumor, and immunosuppressive activities.2-5 They are potent inhibitors of L1210 leukemia cells in vitro, and are also active in vivo against P388 leukemia and B16 melanoma.3 Didemnin B, a more active compound of this class, is approximately twenty times more cytotoxic than didemnin A in vitro and has undergone phase II clinical trials for antitumor activity.3 Both didemnins A and B exhibit antiviral activity against DNA and RNA viruses, with didemnin B being more active.4 The structures of didemnins A and B have been established as 1 and 2, respectively.6 
Structure activity relationship studies have been somewhat limited due to the restricted number of available modifications of the extracted natural compounds. Although the bioactivity of didemnin B has been attributed to its side chain,1b few other structural features have been examined. An X-ray crystal structure of didemnin B by Hossain, et al.,7 shows that the xcex2-turn side chain, the isostatine hydroxyl group, and the tyrosine residue extend outward from the rest of the molecule, leading to speculation about their importance for liological activity. Structural changes in those areas have shown these features to be essential for activity.8 
Although many studies have shed light on the pharmacology and chemistry of didemnins, little is known about their mechanism of action. However, recent biochemical studies of possible binding sites have provided promising results. Studies performed by Shen, et al.,9 have shown that didemnin B binds to a site on Nb2 node lymphoma cells and that this binding may b responsible for the immunosuppressive activity. Schreiber and co-workers10 have reported that didemnin A binds elongation factor 1xcex1 (EF-1xcex1) in a GTP-dependent manner which suggests EF-1xcex1 may be the target responsible for the ability of didemnins to inhibit protein synthesis.
We present here synthetic studies toward a modified macrocycle which possesses an amide bond in place of an ester bond (3). A modification such as, this is likely to result in an increase in hydrogen bonding at the active site, and thus, provide more active compound. In addition, the facile nature of the Cxe2x80x94O bond leads us to believe replacement of these Cxe2x80x94O bonds with Cxe2x80x94N bonds may improve the stability of these compounds.
Synthetic Strategy.
The retrosynthetic disconnections which formed the basis of our plan for the preparation amino-Hip analogue 3 of didemnin A are illustrated in Scheme I. We envisaged disconnection of the amide function between N,Oxe2x80x94Me2-L-tyrosine and L-proline to give the linear heptapeptide 4 and disconnection between L-threonine and isostatine (3S, 4R, 5S) to afford the two units: a tripeptide unit 5 comprised of Nxe2x80x94Me-leucine, threonine, and N,Oxe2x80x94Me2-tyrosine; and a tetrapeptide unit 6 comprised of isostatine, xcex1-xcex1xe2x80x2 amino-isovaleryl) propionyl (Aip), leucine, and proline. 
Synthesis of Tripeptide 5.
Preparation of the diprotected tripeptide unit is shown in Scheme II. Our approach began with methylation of the uncommon amino acid, Cbz-D-leucine, 7, with CH3I/NaH11. Coupling of the derivative Cbz-D-MeLeuOH with the hydroxyl group of the threonine derivative L-TheOEt12 was accomplished with dicyclohexylcarbodiimide (DCC)13 to provide the dipeptide E8. Ester hydrolysis with potassium hydroxide afforded the desired carboxylic acid which was then protected as a phenacyl (Pac) ester 9. Coupling with the tyrosine derivative BocMe2TryOH14 followed by removal of the Boc protecting group15 afforded 10. Removal of the phenacyl function17 provided the key fragment 5. 
Reagents: a) (i) CH3I, NaH, THF; (ii) L-ThrOEt, DCC. CH2Cl2; b) (i) KOH, MeOH; (ii) phenacylBr, Et3N, EtOAc; c) BocMe2TryOH, DCC, DMAP, CH2Cl2; d) Zn, HOAc/H2O.
Synthesis of Tetrapeptide 6.
The construction of fragment 6 involves two novel subunits (2S,4S)-aminoisovalerylpropionic acid (Aip) and (3S, 4R, 5S)-isostatine (Ist). The synthesis of the required isostatine derivative involves (2R, 3S)-allo-isoleucine. The expensive conversion to the hydroxy acid with retention and its conversion in two steps to the amino acid with inversion (Scheme III).18 Conversion of (2S, 3S)-isoleucine to the corresponding xcex1-hydroxy acid 12 was accomplished by using a well-known procedure19 that allows overall retention of configuration via a double inversion. Esterification was carried out with acetyl chloride in methanol, and the corresponding xcex1-hydroxy methyl ester was transformed into the tosloxy methyl ester 13. Treatment of the tosylate with sodium azide in DMF provided the xcex1-azido ester 14 stereoselectively. Saponification of the ester afforded the xcex1-azido acid 15. Hydorgenation of the azide to the free amine proceeded readily in methanol as atmospheric pressure using Pearlman""s catalyst (20% palladium hydroxide on carbon),20 to afford (2S, 3S)-allo-isoleucine 16. 
The major portion of the isostatine subunit, D-allo-isoleucine, 16, was transformed into the tert-butoxycarbonyl (Boc) acid under standard conditions.16 After activation of its carboxyl group as the imidazolide by use of carbonyldiimidazole, treatment with the magnesium enolate of ethyl hydrogen malonate afforded the required xcex2-keto ester 18. The reduction by NaBH4 of the carbonyl group of the xcex2-keto ester was effectively stereospecific, generating the desired (3S, 4R, 5S)-19a as the major product ( greater than 10:1) after chromatographic separation. As shown in Scheme IV, saponification afforded the required Boc-(3S, 4R, 5S)-Ist-OH, 20. 
The next step toward the synthesis of the tripeptide fragment (6) involved formation of the amino Hip subunit. This unit was synthesized from Cbz-L-valine, 21, utilizing a procedure based in part on the work of Nagarajan.21 After activation of its carboxyl group as the imidazolide by use of carbonyl-diimidazole, treatment with the magnesium enolate of ethyl hydrogen methyl malonate (EHMM) afforded the required xcex2-keto ester 22. Sodium borohydride reduction of the xcex2-keto ester produced a diastereomeric mixture of alcohols which were separable by column chromatography. Following saponification and coupling with L-leucine methyl ester (L-LeuOMe), flash chromatography afforded the desired (Pac) bromide provided the protected derivative 24. Oxidation of the secondary alcohol with pyridinium chlorochromate on alumina22 provided the xcex2-keto amide. Removal of the phenacyl protecting group provided the free acid which was coupled with L-proline trimethylsilylester. Catalytic hydrogenation removed the Cbz protecting group and coupling of the isostating subunit 20 with the amine produced the diprotected tetrapeptide. As shown in Scheme V, the Boc protecting group was then removed under standard conditions15 to afford the key tetrapeptide unit 6. 
Reagents: a)(i) CO(imid)2, THF; (ii) EHMM, iPrMgBr; b) (i) NaBH4, EtOH; (ii) KOH, MeOH; (iii) LeuOMe, DCC, CH2Cl2; c) (i) KOH, MeOH; (ii) phenacylBr, Et3N, EtOAc; (iii) PCC, Al2O3, CH2Cl2; d) (i) Zn/HOAc; (ii) ProOTMSe, DCC, CH2Cl2; (iii) H2, Pd/C, MeOH; (iv) BocIstOH 16, DCC, CH2Cl2; (v) HCl, dioxane.
Synthesis of Linear Heptapeptide 4.
The synthesis of the linear heptapeptide 4 involved coupling of the two subunits, Cbz-D-MeLeuThe(OMe2TyrBoc)OH, 5, and H-IstAipLeuProOTMSe, 6. A variety of coupling methods (BopCl,24 DCC,EEDQ25) were attempted, however, the EDCI method26 was shown to be the most efficient for the formation of the triprotected compound 4. Deprotections of the trimethylsilyl ester and the Boc functions were performed under standard conditions to give the monoprotected linear heptapeptide 7. As shown in Scheme VI, cyclization of 7 was achieved by treatment with EDCI to yield the protected compound 3, and catalytic hydrogenation provided the unprotected amino Hip (Aip) analog of didemnin A, 8. 